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The role of Rpd3 and Sir2 in regulation of replication initiation in budding yeast: Rpd3 acts directly on single-copy origins while Sir2 works through ribosomal DNA origins
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The role of Rpd3 and Sir2 in regulation of replication initiation in budding yeast: Rpd3 acts directly on single-copy origins while Sir2 works through ribosomal DNA origins
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Content
THE ROLE OF RPD3 AND SIR2 IN REGULATION
OF REPLICATION INITIATION IN BUDDING
YEAST: RPD3 ACTS DIRECTLY ON SINGLE-COPY
ORIGINS WHILE SIR2 WORKS THROUGH
RIBOSOMAL DNA ORIGINS
by
YIWEI HE
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2016
Copyright 2016 Yiwei He
I
ACKNOWLEDGEMENT
This thesis could not have been realized without a great deal of guidance, and, both
mental and practical, support. I would like to deeply thank those people who,
during the past two years in which this project latest, provided me with everything
I needed.
First of all, I would like to thank my mentor, Dr. Oscar. M. Aparicio, for his
guidance during the entire project. He is a great scientist who is always passionate
in doing research and full of brilliant ideas. I am so glad that I joined this lab and I
learnt a lot from him.
Also, I would like to thank my thesis committee members, Dr. Judd Rice and Dr.
Baruch Frenkel, for their time and guidance.
Nevertheless, I would like to thank each of my lab mates: Dr. Zac Ostrow, Dr.
Jared Peace, Dr. Joanna Haye, Sandra Villwock, Haiyang Zhang and Yan Gan.
They are wonderful people who make Team Aparicio feels like a family.
At last, I would like to thank my friend and family for their love and support.
II
Table of Contents
ACKNOWLEDGEMENT I
List of Tables III
List of Figures IV
ABSTRACT V
INTRODUCTION 1
RESULTS 7
qBrdU-IP-Seq method 7
Rpd3 represses late-firing origins in replication initiation while Sir2 has opposite effect. 9
Rpd3 directly regulates replication initiation at single-copy DNA origins while Sir2 regulates them
through rDNA origins. 15
Fkh1 regulates replication initiation primarily at single-copy DNA origins independently with Rpd3
and Sir2. 17
DISSCUSSION 23
MATERIALS AND METHODS 26
Yeast Strain Constructions 26
Culture Methods 26
BrdU-IP-Seq 27
Analysis of Sequencing Data 28
SUPPLEMENTAL MATERIAL 29
REFERENCES 31
III
List of Tables
Table 1. Strains used in this study.
Table 2. Sir2-sensitive origins.
Table 3. Numbers of origins in different quartiles.
Table 4. Sequencing read counts of rDNA region.
Table 5. Total sequencing read counts.
IV
List of Figures
Figure 1. Preferential recruitment and competing for limiting replication factors as
a timing mechanism.
Figure 2. Rpd3 and Sir2 control the replication initiation at rDNA origin in an
opposite manner.
Figure 3. Stepwise scheme of qBrdU-IP-Seq analysis.
Figure 4. Rpd3 and Sir2 do not significantly affect cell cycle progression.
Figure 5. Validation of the data: the deletion of Rpd3 upregulates BrdU
incorporation signal.
Figure 6. Rpd3 advances initiation timing while Sir2 delays it.
Figure 7. Sir2 significantly advance rDNA origins timing while Rpd3 does not
have directly effect on them.
Figure 8. Fkh1 does not require Rpd3 or Sir2 to reprogram initiation timing in late
G1.
Figure 9. Rpd3 is able to repress origins firing even in the presence of Fkh1.
Figure 10. The scheme of the novel model.
V
ABSTRACT
In budding yeast Saccharomyces cerevisiae, the exact mechanism that regulates
DNA replication timing remains unclear. Previous studies have reported that two
histone deacetylases Rpd3 and Sir2 control replication initiation. In this study, we
investigated Rpd3 and Sir2 in regulation of replication timing genome-wide using
qBrdU-IP-seq. Our results revealed that Rpd3 delays initiation at many of the
single-copy late-firing origins by direct acting on them while Sir2 significantly
represses initiation at rDNA origins and controls single-copy origins timing in an
indirect manner.
Key words: Rpd3, Sir2, Fkh1, rDNA, qBrdU-IP-Seq, replication timing.
1
INTRODUCTION
In eukaryotes, chromosomal DNA replication initiation is governed in a spatially
and temporally co-regulated manner primarily through the coordinated activations
of selective discrete loci, known as replication origins, distributed throughout the
genome. In the budding yeast Saccharomyces cerevisiae, the stepwise initiation of
DNA replication occurs at autonomously replicating sequences (ARS) (Newlon &
Theis, 1993), relatively short (100- to 150- base pair) and considerably A/T rich
chromosomal DNA sequences that consists of a series of modular elements, A, B1,
B2 and B3 (Marahrens & Stillman, 1992, 1994). Among these elements, A element
is the most essential and highly conserved one that contains the 11bp ARS
consensus sequences (ACS)(Broach et al., 1983). ACS together with the adjacent
B1 element directly binds with a six-subunit DNA binding complex termed as
origin recognition complex (ORC) (Rao & Stillman, 1995; Rowley, Cocker,
Harwood, & Diffley, 1995), a central component for DNA replication, that binds
all origins sequences and serves as the foundation for assembly of the pre-
replication complex (Pre-RC) and determines the sites of origins throughout the
cell cycle (Diffley, 2004). This process of pre-RC assembling is required for
subsequent steps in DNA replication initiation and only occurs once per cell cycle
at individual origins. In G1 phase, when cyclin-dependent kinase (CDK) activity
remains low, ORC recruits Cdc6p and Cdt1p together load heterohexameric
minichromosome maintenance (MCM) complex at origins, forming the inactive
form of Pre-RC. Origins with MCM loaded are referred to as “licensed” for
initiation. During the G1 to S transition phase, as CDK level rises, Pre-RC is
activated by the combined action of CDK and Dbf4-dependent kinase (DDK)
(Gambus et al., 2009; Sugino & Araki, 2006). DDK, composed of the catalytic
2
subunit Cdc7 and the regulatory subunit Dbf4, phosphorylates MCM allowing the
recruitment of Cdc45 and Sld3, whereas CDK phosphorylates Sld3 and Sld2
proteins, leading to the recruitment of GINS complex (Labib, 2010). Ultimately,
Cdc45, MCM and GINS forms the active CMG (Cdc45-MCM-GINS) helicase
complex, with additional factors including DNA polymerase to assemble a
complete replisome that stimulates template DNA unwinding and forms two
bidirectional replication forks that initiates the DNA synthesis process (Machida,
Hamlin, & Dutta, 2005). Moreover, the abundance of replication factors, like
Cdc45-Sld3, are limited during replication initiation which allows only a subset of
licensed origins to fire upon S-phase entry and thus late-firing origins are not able
to fire until early-firing origins are terminated and replication factors are recycled
and become available to those unfired-origins (Fig. 1)(O. M. Aparicio, 2013).
Figure 1. Preferential recruitment and competing for limiting replication factors as a timing
mechanism. Limited amount of replication initiation factors is preferentially recruited to early-firing
origins in early G1 phase. During DNA replication, replication forks meet and terminate, allowing
limiting replication factors to be recycled to late-firing origins and subsequently initiated. (O. M.
Aparicio, 2013).
3
Interestingly, although replication origins share some consensus sequence
similarities and follow the same stepwise replication-factor-assembly process for
initiation, a whole-genome view of DNA replication shows different origin
activities and origins fire in distinct timing patterns. The involved mechanism
remains unclear, however, it is commonly believed that replication origin timing is
influenced by chromatin structure and sub-nuclear localization. In general, origins
within heterochromatic and peripherally located regions, such as telomeric and
subtelomeric regions, have lower accessibility to replication factors and thus are
typically late-replicating, whereas origins near centromere fire earlier than other
locations (O. M. Aparicio, 2013; Rhind & Gilbert, 2013; Smith & Aladjem, 2014).
Histone deacetylases (HDACs), a type of chromatin remodelers, are a class of
enzymes that remove acetyl groups from the histone core of the nucleosome,
allowing histones to be wrapped with the chromatin more tightly and thus forming
more condensed chromatin structure that is less favorable to replication factors. In
budding yeast, Rpd3 has been found to act as a histone deacetylase that regulates
transcription, gene silencing and other processes by influencing chromatin
remodeling (Ruiz-Roig, Vieitez, Posas, & de Nadal, 2010; Rundlett et al., 1996; Yi
et al., 2012). In more detail, Rpd3 together with Sin3 is able to forms two
functionally distinct Rpd3 complexes with different subunits. Large Rpd3 complex
(Rpd3L), acting as a transcriptional regulator, is recruited by sequence-specific
DNA-binding proteins to promoters that represses gene expression by
deacetylating proximal histones (Kadosh & Struhl, 1997; Rundlett et al., 1996);
While small Rpd3 complex (Rpd3S) is nonspecifically recruited to transcribed
open reading frames to suppress transcription initiation (Carrozza et al., 2005;
Joshi & Struhl, 2005). In addition, Sin3–Rpd3 may also affect replication timing
4
through Rpd3L’s function as a promoter specific regulator of gene expression, and
through Rpd3S’s function in condensing chromatin within transcribed regions that
flank origins in the proximal intergenic regions. In fact, Rpd3 has been suggested
playing a role in replication initiation timing that causes delayed initiation of many
non-telomeric and late-firing origins, along with increased acetylation of histones
flanking these origins (J. G. Aparicio, Viggiani, Gibson, & Aparicio, 2004; Knott,
Viggiani, Tavare, & Aparicio, 2009; V ogelauer, Rubbi, Lucas, Brewer, &
Grunstein, 2002).
Another histone deacetylase Sir2, best known for its role in the formation of
budding yeast heterochromatin at telomeres and the cryptic mating-type loci HMR
and HML, has been reported to negatively regulates origin activity at the level of
pre-RC assembly (Fox & McConnell, 2005; Gilson & Geli, 2007; Rusche,
Kirchmaier, & Rine, 2003). Sir2 might inhibit Pre-RC assembly by the
deacetylation of Cdc6 and further prevent MCM recruitment or through transient
sequence-specific recruitment to an inhibitory sequence I
s
that was identified at the
3’ of sir2-sensitive origins to affect chromatin structure. It is also possible that sir2
deacetylates H4K16 that is adjacent or overlapping B2 element within ARS at a
low level throughout the genome which potentially affects the initiation timing
globally (Crampton, Chang, Pappas, Frisch, & Weinreich, 2008).
It has also been reported that Sir2 and Rpd3 control replication timing through
their opposite effects on ribosomal DNA (rDNA) origins initiation. rDNA origins
are located within repetitive DNA sequences that take up one-third of the budding
yeast genome. Normally, rDNA locus consists of 150 to 200 copies of a 9.1 kb
repeating unit, organized in a single tandem array on chromosome XII. Each unit
contains an ARS element for replication initiation, but only 20% of these origins
5
fire in S phase (Brewer & Fangman, 1988). In terms of replication timing
regulation, Sir2 represses initiation at rDNA origins, whereas Rpd3 counteracts this
effect. In addition, previous study hypothesized that repetitive DNA origins, like
rDNA origins, competes with single-copy origins for limiting initiation factors to
fire indicating that Sir2 and Rpd3 could regulate replication initiation globally in
an indirect manner (Figure 2).
Figure 2. Rpd3 and Sir2 control the replication initiation at rDNA origin in an opposite manner. In
wildtype (WT), Sir2 represses initiation at rDNA origins while Rpd3 counteracts this effect. Limiting
initiation factors are relatively evenly distributed between rDNA origins and single-copy origins. In sir2∆
cells, rDNA silencing is downregulated, increasing origin accessibility to limiting initiation factors and
thus advances rDNA origins timing; The loss of Rpd3, however, upregulates rDNA silencing and thus
represses rDNA origins initiation. (Yoshida et al., 2014)
6
In budding yeast Saccharomyces cerevisiae, forkhead DNA binding proteins Fkh1
and Fkh2 are highly evolutionary conserved and have been characterized largely
with respect to their function as transcription factors for mitotic cyclin CLB2
cluster (Murakami, Aiba, Nakanishi, & Murakami-Tonami, 2010). In contrast to
histone deacetylase, which affects most of the late-firing origins, Fkh1 and Fkh2
are rate-limiting replication origin activators of most non-telemetric, early-firing
origins in the genome (Knott et al., 2012). It is thought that Fkh1 and Fkh2
promote early replication through the clustering of origins and thus increase the
accessibility to limiting initiation factors, like Cdc45 (Smith & Aladjem, 2014).
Nevertheless, studies show that both of the Rpd3 and Sir2 are associated with Fkh1
and Fkh2 in regulation of gene transcription. For example, Fkh1 and Fkh2 require
Rpd3L to act as selective anti-activators, preventing Swi5 but not Ace2, from
activating (Voth et al., 2007); Fkh1 and Fkh2 associate with Sir2 in G1 and M
phase, and Sir2 regulates the expression of CLB2 gene through Fkh1/Fkh2-
mediated binding to the CLB2 promoter (Linke, Klipp, Lehrach, Barberis, &
Krobitsch, 2013). However, the regulatory relationships between Rpd3 and/or Sir2
and Fkh1 in replication remain vague.
In this study, we explored the regulatory role of Rpd3 and Sir2 on replication
initiation genome-wide and also specifically on rDNA origins. We tested whether
rDNA origins initiation would compete with that of single-copy origins. Lastly, we
investigated whether fkh1 functionally correlates with Rpd3 and/or Sir2 in the
regulation of replication initiation.
7
RESULTS
qBrdU-IP-Seq method
To examine the genome-wide DNA replication profiles and make quantitative
comparisons of origin firing between strains, our lab has previously developed the
method quantitative BrdU immunoprecipitation analyzed by high-throughput DNA
sequencing (BrdU-IP-seq) (Peace, Villwock, Zeytounian, Gan, & Aparicio, 2016).
5’-bromo-2’-deoxyuridine (BrdU) is an analog of thymidine that can be
incorporated into newly synthesized DNA of replicating cells. In this method, in
order to facilitate the cells to assimilate BrdU from the growth medium, we
constructed BrdU-incorporating (BrdU-inc) strains (Viggiani & Aparicio, 2006).
To synchronize BrdU-inc strains, MAT-a-type cells are first arrested in late G1
phase with α-factor for at least one doubling-time and then released into α-factor
free fresh growth medium in the presence of hydroxyurea (HU), an inhibitor of
ribonucleotide reductase, which starves DNA polymerases by depleting dNTPs and
thus arrests cells in early S-phase after early origin firing while inhibiting late or
dormant origins through intra-S checkpoint signaling (Santocanale, Corrado, and
John FX Diffley. Nature 395.6702 (1998): 615-618.). Cells were harvested after
completely releasing from S phase and the BrdU-labeled genomic DNA was
extracted and randomly sheared into small fragments around 300bp. Equal amount
of the genomic DNA from each of the samples is uniquely barcoded by end-
ligation of unique Illumina-compatible linkers.
In the qBrdU-IP-seq method, multiple uniquely barcoded samples are pooled
together to make the DNA library prior to the BrdU-IP and library amplification
steps and a small fraction of this pool is set aside as “input/total,” while the
8
remainder is immunoprecipitated by anti-BrdU antibody. Both of the
immunoprecipitated and total DNA are then polymerase chain reaction (PCR)
amplified with indexed primers and sequenced on Illumina instrument. IP DNA
sequencing data are then normalized against total DNA sequencing data, and thus,
those potential DNA manipulations for library preparation, addition of adapters,
which include the barcodes, is carried out prior to depletion of DNA quantity by
IP; and numerous operations that may introduce substantial variability between
individually prepared samples, including IP, PCR amplification, and quantification
of sequencing reads, are conducted under identical and/or more reliable conditions
(Fig. 3). Our previous studies and also this study show that the qBrdU-IP-Seq
method is valid to accurately quantify and directly compare replication levels at
origins among different strains or different experimental conditions.
Figure 3. Stepwise scheme of qBrdU-IP-Seq analysis. BrdU-labeled genomic DNA from each sample
is barcoded by end-ligation of Illumina-compatible linkers. Samples are pooled, a small fraction of this
pool is set aside as “Input,” and the remainder is subjected to immunoprecipitation (IP) with anti-BrdU
antibody. The IP and Input samples are PCR-amplified with indexed primers and sequenced. IP sample
reads are normalized against Input sample reads (Peace et al., 2016).
9
Rpd3 represses late-firing origins in replication initiation while Sir2 has
opposite effect.
In budding yeast Saccharomyces cerevisiae, the exact mechanism that regulates
replication initiation timing has remained vague, however, it is clear that chromatin
modification plays an essential role. For example, histone deacetylation allows the
assembly of a more condensed chromatin structure that decreases the accessibility
of replication initiation factors within the region and thus delays initiation timing.
Previous study showed that the loss of histone deacetylase Rpd3 result in higher
levels of histone H3 and H4 acetylation surrounding Rpd3-regulated origins and
the alterations of chromatin structure is likely responsible for the advanced
initiation timing of normally late-firing origins. BrdU-IP-chip analysis identified
104 late-firing replication origins that are regulated by Rpd3, which is specifically
targeted to promoters to silence (Knott et al., 2009).
In a different study, plasmid loss data showed that deletion of histone deacetylase
Sir2 rescued the growth defect of cdc6-mutant cells suggesting it is possible that
Sir2 inhibits Pre-RC assembly through the repression of Cdc6 binding and further
prevent MCM recruitment, namely, origin licensing for replication initiation. In
addition, consensus sequence analysis revealed similarity among those Sir2-
sensitive origins and an essential sequence has been identified from them and
termed as I
s
element which is thought to be required for Sir2 to function. This leads
to another possible mechanism that Sir2 inhibits origins for firing by the specific
recognition and binding to I
s
element. Nevertheless, sir2 deacetylates H4K16 that
is adjacent or overlapping B2 element within ARS at a low level throughout the
genome which potentially affects the initiation timing globally (Crampton et al.,
2008; Fox & Weinreich, 2008).
10
To examine the regulatory role of Rpd3 and Sir2 in DNA replication on a genome-
wide scale, we began with DNA content analysis of WT, rpd3∆, sir2∆ and
sir2∆rpd3∆ throughout an unperturbed, synchronous S phase. Cells were first
grown in YEP-glucose into log-phase (OD
600
~0.5) and synchronized in late G1
phase by incubation with α-factor for 4 hrs. Cells were then released into S phase
by incubation in fresh medium. The time point of release was treated as “0min”
and aliquots were harvested at 15min intervals for 90min in total. DNA contents
were measured by fluorescence-activated cell scanning (FACS).
Figure 4. Rpd3 and Sir2 do not significantly affect cell cycle progression. DNA content analysis of
strains CVy43 (WT), CVy44 (rpd3∆), YWy3 (sir2∆) and YWy6 (sir2∆rpd3∆) grown in YEP- glucose,
blocked in G1 phase by incubation with α-factor for 4 hrs. Cells were released from G1 block into S
phase by incubation with Pronase E in fresh YEP-glucose without α-factor and aliquots were harvested at
indicated time intervals. Data show no significant differences of cell cycle progression among
experimental strains.
FACS data shows that the S phase completes slightly sooner in rpd3∆ cells than in
WT cells, while sir2∆ and sir2∆rpd3∆ cells are very similar to WT, indicating that
neither Rpd3 or Sir2 significantly affects cell cycle progression (Fig. 4).
11
To generate genome-wide replication profiles in HU, we applied qBrdU-Seq to
above strains. BrdU-IP-Seq was done by cell cycle block-and-release as indicated
above except that cells were released into early S phase in the presence of BrdU as
well as HU, the latter allowing early but not late/dormant origins to initiate
replication. Cells were harvested 60min after release as indicated in previous DNA
content analysis. To validate the data, we compared BrdU incorporation signals
among experimental strains at previous identified Rpd3-regulated origins. Heatmap
is generated presenting the average BrdU incorporation signal for 5-kb regions
centered on overlapping Rpd3-regulated origins. This result shows ~2 folds
increased BrdU signal in rpd3∆, which is constant with our previous observation.
Figure 5. Validation of the data: the deletion of Rpd3 upregulates BrdU incorporation signal. BrdU
incorporation signal was averaged across 5-kb regions aligned by the peak summit of Rpd3-regulated
origins.
Chromosomal plots of the qBrdU-seq data show that in WT cells, BrdU
incorporation level is high at early origins, while its signal at later-firing origins is
substantially lower, as expected. In rpd3∆ cells, stronger BrdU signal is detected
among late-firing origins compared to those in WT, indicating the loss of Rpd3
increases activation of many later-firing origins relative to earlier-firing origins. In
12
contrast, the initiation of replication is significantly reduced at both of the earlier-
and later-firing origins in sir2∆ cells and it loses BrdU peaks at many chromosomal
regions where BrdU signal is detectable in other strains. Interestingly, sir2∆rpd3∆
cells show similar pattern of DNA replication initiation profile as WT with
generally weaker BrdU peaks than those in WT cells, suggesting that the deletion
of Rpd3 reverses the effect of SIR2 deletion. For example, plot of BrdU
incorporation along Chromosome XI shows four major peaks (early origins) and
several minor peaks (late/inefficient origins) in WT cells, whereas rpd3∆ shows
greater BrdU incorporation at virtually all of the minor peak positions in addition
to some origin loci lacking BrdU incorporation in WT cells, in sir2∆ cells, almost
all of the minor peaks in WT are gone and the BrdU level of the remaining major
peaks are relatively lower than in WT. At last, sir2∆rpd3∆ shows similar BrdU
incorporation pattern as WT with generally weaker signals (Fig. 6A).
The results of Weinreich’s lab suggest that Sir2 delays replication initiation timing
by inhibiting Pre-RC assembly at a subset of origins.
Thus we examined the replication initiation profile at Sir2-sensitive origins that
have been identified previously (Table. 2). Plot of BrdU incorporation along
Chromosome III shows no distinguishable differences among experimental strains
at ARS305, ARS315 and ARS317 (marked with dash lines) (Fig. 6A), indicating
Sir2 does not seem like inhibit Pre-RC assembly at those origins. However, it is
also possible that this negative result is due to the less sensitivity of qBrdU method
compare to the extremely sensitive plasmid loss assay applied in Weinreich’s lab.
13
Figure 6. Rpd3 advances initiation timing while Sir2 delays it. (A) qBrdU-seq analysis of CVy43
(WT), CVy44 (rpd3∆), YWy3 (sir2∆) and YWy6 (sir2∆rpd3∆). Cells grown in YEP-glucose were blocked
in G1 phase by incubation with α-factor for 4hs. Cells were released from G1 block into S phase by
incubation in fresh YEP-glucose, minus α-factor, plus Pronase E, BrdU and HU for 1 h and harvested.
Experiment was performed in triplicate and averaged data plotted for BrdU peaks along chromosome III
and XI with underneath dots presenting Fkh-regulated origins, dash lines presenting Sir2-sensitive
origins. (B) Origins were divided into quartiles according to T
rep
. Bar plot and pie chart are presenting the
numbers and proportion of the MACS called origins. (C) Venn diagrams of all origins identified in the
indicated sets. (D) Origins were divided into quartiles and rDNA origin,
and BrdU incorporation was
averaged across 5-kb regions aligned by the peak summit of each origin.
14
To characterize the effects of Sir2 and Rpd3 across the genome, we determined
whether the level of BrdU incorporation at replication origins was significantly
altered. BrdU incorporation peaks were called using Model-based Analysis of
ChIP-Seq (MACS) and were compared with confirmed/likely origins on OriDB to
filter out non-origin peaks. The Venn-diagram shows 235 single-copy origins firing
in WT cells, 301 firing in rpd3∆ cells, 192 firing in sir2∆ and 226 firing in
sir2∆rpd3∆ cells, respectively, which is consent with the BrdU incorporation plots.
Most of these origins overlap among all groups with 65 additional origins detected
in rpd3∆ cells and 36 origins missing in sir2∆ cells (Table. 3; Fig. 6B, C). To be
more detailed, origins were divided into quartiles based on their replication timings
(T
Rep
). The result shows that in rpd3∆ cells, there is a decreased proportion of
origins in the first quartile (early origins) and an increased proportion in other
quartiles (late origins) compared to WT, indicating that the extra origins fired in
rpd3∆ cells are late-firing origins. In contrast to rpd3∆, there is an increased
proportion of origins in the first quartile in sir2∆ cells and a decreased proportion
in other quartiles, especially in the second quartile, suggesting the missing origins
in sir2∆ are likely from that category (Fig. 6B).
In order to examine genome-wide replication initiation among those origin groups,
we plotted the average BrdU incorporation signal for 5-kb regions centered on
origins. The result shows that WT has the strongest signal in the first quartile,
rpd3∆ has the strongest signal in the second, third, and fourth quartiles, sir2∆ cells
have the weakest signal of all quartiles and sir2∆rpd3∆ cells have intermediate
signal levels that are close to sir2∆ (not really in early quartiles). This result is
consistent with previous conclusions that Rpd3 affects most of the late-firing
origins and Sir2 limits origin firing on a large scale (Fig 6D).
15
Together, these data demonstrate that Rpd3 represses replication initiation at many
late-firing origins rather than early-firing origins. Sir2 appears to advance origin
firing genome-wide.
Rpd3 directly regulates replication initiation at single-copy DNA origins while
Sir2 regulates them through rDNA origins.
Most origins in budding yeast genome are located in single-copy DNA regions
such as those origins analyzed above. However, about one-third of the origins are
located within repetitive DNA sequences, like rDNA array. rDNA locus normally
consists of 150 to 200 copies of a 9.1kb repeat, organized in a single tandem array
on chromosome XII. Each of the repeat contains an ARS element and
approximately 20% of these origins fire in a given S phase. Recent study
hypothesized that during replication initiation, rDNA origins compete with single-
copy DNA origins for firing and Sir2 plays an essential role in silencing rDNA
origins whereas Rpd3 has an opposite effect.
To determine the role of Rpd3 and Sir2 on rDNA origins initiation and whether the
alteration of rDNA origins firing would compensate that of single-copy DNA
origins firing in overall BrdU incorporation level, we compared the level of rDNA
origin firing among WT, rpd3∆, sir2∆ and sir2∆rpd3∆ strains. To accomplish that,
we took advantage of the “input/total” DNA, for the reason that, unlike IP DNA
sequencing reads, they are the original genome sequencing reads without BrdU
immunoprecipitation and enrichment. The sum of sequencing reads from both of
the whole genome and within rDNA region were generated and then calculated the
ratio between them (Table. 4). Data show that there is no distinguishable difference
of rDNA copy number amount all strains and they roughly contain 5% of rDNA
out of the whole genome (Fig. 7A). Total BrdU incorporation signal from each of
16
the mutant strain was normalized by WT and data show that out of all the BrdU-
incorporation signal detected from the whole genome, 18% derives from rDNA
origins and 82% from single-copy origins in WT; In rpd3∆, rDNA origins firing is
repressed with only 3% of the total signal and almost all the BrdU signal is
contributed by single-copy origins; In contrast to rpd3∆, sir2∆ has ~2-fold increase
of BrdU signal from rDNA compared to WT, however, the overall BrdU signal
level is lower than WT; Surprisingly, sir2∆rpd3∆ has very similar rDNA origins
firing strength as sir2∆ with increased single-copy origins firing which makes the
total BrdU signal comparable to WT (Table. 5; Fig. 7B). By comparing the
averaged BrdU signal at single-copy origins and rDNA origins, it seems that in
rpd3∆, the advancement of late-firing single-copy origins significantly repress
rDNA origin firing whereas the downregulated firing signal at single-copy origins
in sir2∆ is largely due to the dramatically upregulated at rDNA origin. Again,
sir2∆rpd3∆ have undistinguishable BrdU signal on rDNA origins with sir2∆,
indicating the further deletion of Rpd3 in sir2∆ does not make much changes on
rDNA origins. Also, the BrdU incorporation level on rDNA is comparable to the
second quartile single-copy origins, suggesting rDNA origins are relatively early
origins by default (Fig 6D).
Figure 7. Sir2 significantly advance rDNA origins timing while Rpd3 does not have directly effect
on them. (A) The ration of rDNA region out of the whole genome. (B) Total BrdU signal of single-copy
17
origins versus rDNA origins.
Together, these data suggest that Rpd3 has a direct repressive effect on late-firing
single-copy DNA origins and plays either no effect on rDNA origins or on different
pathways from Sir2 when regulating rDNA origins. And the hypothesis that single-
copy DNA origins compete with rDNA origins for replication initiation can explain
how the advanced timing of most late-firing single-copy origins in rpd3∆ cells
would thus limit rDNA origins firing. In contrast, Sir2 advances the replication
initiation on early-firing single-copy origins through its directly inhibitory role on
rDNA origin initiation. The loss of Sir2 causes more initiation to occur on single-
copy DNA origins in the first quartile of origins, with smaller numbers of origins
firing in the second quartile, suggesting rDNA origins is able to compete with the
second quartile origins but not those earliest origins, which constant with the
increasing number in the first quartile decreasing number in the second quartile in
sir2∆ in Figure 6B. However, since rDNA origins only takes one-third out of the
total numbers of origins, the increasing initiation of rDNA origins is not able to
compensate the decrease of single-copy origins in the sense of overall effect.
Fkh1 regulates replication initiation primarily at single-copy DNA origins
independently with Rpd3 and Sir2.
In budding yeast Saccharomyces cerevisiae, Forkhead Box (FOX) DNA binding
proteins Fkh1 and Fkh2 have been well characterized as transcription factors that
regulate the of expression of the CLB2 cluster of genes during G2/M phase
(Murakami et al., 2010). Later study has demonstrated that Fkh1 and Fkh2 are also
responsible for stimulating the initiation of most non-centromeric, early-firing
origins in the yeast genome (Knott et al. 2012). In the absence of Fkh1 and Fkh2,
many of the early-firing origins are significantly delayed in replication initiation
18
timing and thus defined as “Fkh-activated” origins, while a relatively smaller
group of normally later-firing origins are advanced in activation and thus defined
as “Fkh-repressed” origins. The exact mechanism of the replication initiation
regulated by Fkh1 and Fkh2 remains unclear but is thought that they establish early
replication timing at Forkhead-activated origins by directly acting in cis to recruit
these origins into clusters where limiting replication factors are concentrated.
Recent study has showed that both Rpd3 and Sir2 are associated with Fkh1 and
Fkh2 in regulation of the gene expression (Linke et al., 2013; Pondugula et al.,
2009; Voth et al., 2007), however, their co-regulatory role in stimulating replication
initiation have not been fully explored.
In order to examine whether Rpd3 and Sir2 functionally correlates with Fkh1 in
replication initiation, we examined the effect of RPD3 and SIR2 deletion on DNA
origins classified in the previous study as “Fkh1-activated”, “Fkh1-repressed” and
“Fkh1-unregulated”. Previous BrdU incorporation plot along Chromosome III and
XI shows that rpd3∆ has higher BrdU peaks than WT at all of the Fkh1-repressed
origins while sir2∆ has lower BrdU peaks at almost all origins (Fig. 6A)
19
Figure 8. Fkh1 does not require Rpd3 or Sir2 to reprogram initiation timing in late G1. (A) qBrdU-
seq analysis of YWy4 (OE-Fkh1 in WT), YWy5 (OE-Fkh1 in rpd3∆) and YWy6 (OE-Fkh1 in sir2∆).
20
Cells grown in YEP-raffinose (noninducing) were blocked in G1 phase by incubation with α-factor for
3hrs and resuspended in YEP-galactose plus α-factor for 2 hrs to induce Fkh1 expression while
maintaining in G1 block. Cells were released from G1 block into S phase by incubation in fresh YEP-
galactose, minus α-factor, plus Pronase E, BrdU and HU for 1 h and harvested in 90min. Experiment was
performed in triplicate and averaged data plotted for BrdU peaks along chromosome III and XI with
underneath dots presenting OE-Fkh1-regulated origins. (B) Bar plot shows MACS called origins. (C)
Venn diagrams of all origins identified in the indicated sets. (D)(E) BrdU incorporation was averaged
across 5-kb regions aligned by the peak summit of indicated origin. (F) Origins were divided into
quartiles and rDNA origin,
and BrdU incorporation was averaged across 5-kb regions aligned by the peak
summit of each origin.
Next, in order to test whether the over-expression of Fkh1 would exhibit a
profound change in replication profiles of mutant strains in HU, we transformed a
plasmid vector with FKH1 expression under control of the GAL promoter into WT,
rpd3∆ and sir2∆ cells. These cells were synchronized in G1 phase in non-induced
medium YEP+2% raffinose and then shifted to YEP+2% galactose to induce Fkh1
expression for 2 hours and harvested in 90min after releasing. Chromosomal plots
of the qBrdU-Seq data show relatively more similar BrdU peak patterns among
OE-Fkh1 group than non-OE-Fkh1 group in the previous, but still with more minor
peaks in OE-Fkh1 rpd3 and generally smaller peaks in OE-Fkh1 sir2∆. For
example, plot of BrdU incorporation along Chromosome XI shows reduced BrdU
incorporation in OE-Fkh1 sir2∆ at most of the OE-Fkh1 activated origins while
Chromosome III shows non-distinguishable differences at OE-Fkh1 repressed and
un-regulated origins among all experimental strains (Fig.8A). Genome-wide view
of the replication profile in HU shows that there are 238 origins detected in OE-
Fkh1 WT, 312 in OE-Fkh1 rpd3∆ and 153 in OE-Fkh1 sir2∆, slightly increasing
number of origins in first two groups but a dramatically decreasing number of
origins in sir2∆ compared with non-over-expression of Fkh1 group (Fig. 8B, C).
To examine the BrdU incorporation level along genome, we generated the
heatmaps presenting the average BrdU incorporation signal for 5-kb regions for
21
both of the non-overexpression group and OE-Fkh1 group centered on overlapping
Fkh-regulated or OE-Fkh1-regulated origins. Data show that overexpress of Fkh1
reprogramed the initiation timing in late G1 result in decreased differential BrdU
signal among experimental strains (Fig8D, E). Finally and surprisingly, BrdU
signals reach at pretty much the same level on all single-copy origins and rDNA
origins when overexpress Fkh1 in WT and mutant strains. These results suggest
that Fkh1 is a fundamental regulator in replication initiation and it does not require
neither Rpd3 or Sir2 to reprograms the timing in late G1. Furthermore, it is
possible that Fkh1 recognizes only single-copy origins but not rDNA origins and
abundant Fkh1 would help those single-copy origins compete with rDNA origins
for firing. In rpd3∆ cells, overexpression of Fkh1 additionally advances late-firing
origins initiation timing which causes way more single-copy origins join into the
competition and thus allow some of the rDNA origin firing. Whereas,
overexpression of Fkh1 allows those repressed single-copy origin to fire in a
relatively normal level and thus competes with rDNA origins for firing and lowers
the BrdU incorporation level on rDNA origins.
At last, in order to examine whether Rpd3 plays a profound role in preventing late-
firing origins for firing even in the presence of Fkh1, we compared the BrdU
incorporation signal among OE-Fkh1 group. Heatmap shows that there is an
upregulation of replication initiation in OE-Fkh1 rpd3∆ cells, indicating that Rpd3
is able to delay late-firing origins timing even there is abundant Fkh1 around. This
result suggests that Rpd3 might affect different groups of origins from Fkh1.
22
Figure 9. Rpd3 is able to repress origins firing even in the presence of Fkh1. BrdU incorporation was
averaged across 5-kb regions aligned by the peak summit of indicated origin.
23
DISSCUSSION
DNA replication is a vital biological process in all living organisms that is tightly
regulated to preserve genetic information and cell functionality. However, the
faithful and timely replication of the genome can be challenged by the
malfunctioned regulators. The regulation failure of DNA replication result in too
few/too many origins firing at the same time or uncoordinated origins firing which
are the main causes of a transient slowing or stalling of the replication fork that is
defined as “replicative stress” (Magdalou, Lopez, Pasero, & Lambert, 2014).
Importantly, replication stress has been characterized as a major source of the
genomic instability for the reason that it can lead to replication fork collapse,
incompletely replicated DNA, DNA double strand breaks, and replication-
transcription conflict that further lead to cancer and other diseases (Boyer, Walter,
& Sorensen, 2016).
Rpd3 and Sir2 are homologous to class I and III HDAC family in mammalian cells
that are able to regulate DNA replication initiation in yeast (Haberland,
Montgomery, & Olson, 2009). In this study, we demonstrated that Rpd3 directly
inhibits replication initiation primarily at a majority of late-firing origins, while
does not seem to have a strong effect on early-firing origins. This finding supports
our previous conclusion that Rpd3 delays replication timing at many late-firing
origins. Also, our results suggest that Rpd3 does not seem to affect rDNA origins
timing, at least it requires Sir2 to fulfill its function, which argues the hypothesis
suggested in Yoshida’s lab that Rpd3 plays opposite role with Sir2 in rDNA
origins and directly advances their firing. Moreover, although Fkh1 is an essential
regulator in advancing replication timing globally, Rpd3 is still able to repress
replication initiation in a large scale, indicating that Fkh1 and Rpd3 affect different
groups of origins which potentially support the result that there are no Fkh1
24
binding sites flanking Rpd3-regulated origins. While Rpd3 has a direct effect on
single-copy origins, Sir2 appears to play no or subtle role in directly regulating of
those origins. Instead, Sir2 significantly repress rDNA origins replication initiation
and regulate its timing genome-widely through the competition for limiting
initiation factors between single-copy origins and rDNA origins (Fig.10). Previous
studies have characterized Fkh1 as a rate-limiting activator in regulation of
replication timing. This study further demonstrates that over-expression Fkh1 in
late G1 phase reprograms replication timing and it does not require either Rpd3 or
Sir2 for Fkh1 to advance early-firing origins initiation. Also, Fkh1 appears to only
recognize single-copy origins but not rDNA origins and helps single-copy origins
to compete with rDNA origins for initiation, which constant with the previous
result that overexpression of Fkh1 in WT decreased rDNA origins firing.
In summary, the loss of Rpd3 leads to too many origins firing in an early state and
the loss of Sir2 causes more rDNA origin firing which result in an unbalanced
replication initiation between rDNA origins and single-copy origins. Both of the
consequences are potential causes of human diseases that needs further studies.
25
Figure 10. The scheme of the novel model. Rpd3 directly represses single-copy origins and Sir2 silences
rDNA origins. Fkh1 recognizes single-copy origins and advances their timing. The distribution of limiting
initiation factors is influenced by these regulators.
26
MATERIALS AND METHODS
Yeast Strain Constructions
All yeast strains involved in this study are congenic with W303 and are derived
from BrdU-incorporating strains CVy43, MATa ade2-1 ura3-1 his3-11,15 trp1-
1leu2-3,112 can1-100 RAD5+ bar1::hisG BrdUInc::URA or CVy61 MATa ade2-1
ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 RAD5+ bar1::hisG BrdUInc::TRP
(Viggiani & Aparicio, 2006). CVy44(rpd3Δ::KanMX), YWy3(sir2Δ::KanMX) and
YWy6 (sir2Δ::KanMX rpd3Δ::His) were constructed by deletion of SIR2 and or
RPD3 in one of the two wild-type strains mentioned above by PCR amplifying
gene deletion cassette and through long oligonucleotide-based homologous
replacement into the genome and selection for corresponding marker.
For the reason that SIR2 plays a crucial role in silencing at mating type cassette
HML and HMR locus, sir2Δ strains had had problem with responding to a-factor,
HMLɑ locus had been further deleted in all sir2Δ strains.
Plasmid p415-GAL-Fkh1 were introduced into WT, sir2 and rpd3 background
respectively by lithium acetate transformation and selection of appropriate drop-off
medium (Gietz & Schiestl, 2007) for the purpose of over-expression of FKH1.
Culture Methods
For G1-phase block-and-release, cells were inoculated into YEP+2% glucose at
25 ℃, grown to mid-log phase (at O.D. ~0.5) and blocked in G1 by incubation with
7.5nM a-factor at 25 ℃ for 3hrs and added half-dose ɑ-factor for one more hour.
Arrested cultures were checked under microscope and released from G1-phase
arrest by resuspension in fresh YEPD at O.D. 1.0 with 200µg/mL Pronase E
(Sigma-Aldrich, P5147) and gentle sonication to disperse cells. For early S-phase
analysis of replication, cells were released into the presence of 0.2M HU (Sigma-
27
Aldrich, H8627) and 400µg/mL BrdU for 60min at 25 ℃. For the time-course
DNA content analysis, cells were fixed with 70% ethanol overnight, washed, and
resuspended in 50mM sodium citrate, pH7.4, and RNase A was added to 0.2mg/ml
and incubated for 3 h at 50°C. Proteinase K was added to 0.5mg/ml and incubated
50°C for 2h, after which SYTOX Green (Molecular Probes) was added to 1µM for
at least 30min before analysis on a FACScan instrument (Zhong et al., 2013).
For late G1 block, induction, and release, cells were preselected on DOB-Leu for
plasmid and then were inoculated into YEP+2% raffinose at 25°C, grown to mid-
log phase, and blocked in G1 as mentioned above. For induction, cells were
pelleted and resuspended in YEP+2% galactose+7.5 nM α-factor at 25°C for 2 h,
and cultures were released into S phase as mentioned above.
BrdU-IP-Seq
Genomic DNA was isolated from 20mL BrdU-labled cultures as described
previously (Viggiani, Knott, & Aparicio, 2010).Genomic DNA was sheared to
~300bp average using a Covaris S2 instrument. Sheared DNA was end-repaired
and ligated with a barcoded, Illumina-compatible adapter using the KAPA
Biosystems Hyper prep kit(KR0961). Adapter-ligated genomic DNA was purified
and its concentration was determined by spectroscopy (Nanodrop). Equal amounts
of multiple such barcoded DNA samples around 500ng were pooled together; 5%
of the pooled DNA were set aside as “Input” and 95% of that was subject to BrdU-
IP as described previously. The IP and Input DNA samples were PCR-amplified
separately with indexed Illumina-compatible primers. Amplified DNA was isolated
using AMPure beads (Beckman Coulter, A63880) and validated and quantified on
a Bioanalyzer (Agilent), and pooled. DNA sequencing (50 bp paired-end) was
performed on a NextSeq instrument (Illumina) by the NextGen Sequencing Core of
the USC Norris Cancer Center.
28
Analysis of Sequencing Data
Barcodes were split using the barcode splitter ea-utils. Sequence libraries were
aligned to S. cerevisiae genome release r.64 using Bowtie2. The first 10 bp were
trimmed from the 5’ end to account for the barcode and allow for proper
alignment. Aligned sequences were sorted and binned into 50 bp non-overlapping
bins(Li et al. 2009; Quinlan and Hall 2010), normalized by dividing each IP bin by
its corresponding Input bin, yielding an IP:Input ratio, which was median-
smoothed over a 500- or 2000-bp window for HU experiments. Experimental
replicates were averaged and data were arbitrarily scaled, setting “1” for the
99.995% maximum (to get rid of the outliers) average WT signal by dividing all
samples within a qBrdU-seq pool by the same value (i.e., the 99.995% maximum
average WT signal). BrdU peaks were called using MACS 1.4.2 with nomodel
mode (p,0.01). Called peaks were then cross-referenced against origins defined in
OriDB as ‘‘confirmed’’ or ‘‘likely’’ to eliminate any peaks not aligning with an
origin. Overlapping origins among different peak sets were determined by bedtools
using intersect function.
29
SUPPLEMENTAL MATERIAL
Table 1. Strains used in this study.
Table 2. Sir2-sensitive origins.
Strain Name Genotype Mating Type
CVy43 BrdU-inc::URA a
CVy44 rpd3Δ::KanMX, BrdU-inc::URA a
CVy61 BrdU-inc::TRP a
YWy3
sir2Δ::KanMX, HMLɑΔ::URA, BrdU-
inc::TRP a
YWy4
over-expression of Fkh1, sir2Δ::KanMX,
HMLɑΔ::URA, BrdU-inc::TRP a
YWy5
over-expression of Fkh1, rpd3Δ::KanMX,
BrdU-inc::URA a
YWy6
sir2Δ::KanMX, rpd3Δ::HIS,
HMLɑΔ::URA, BrdU-inc::TRP a
YWy7 over-expression of Fkh1, BrdU-inc::URA a
Name Chromosome Start End Trep (min)
ARS305 III 39158 39706 12.64
ARS315 III 224807 225053 15.19
ARS317 III 292524 292826 46.61
ARS603 VI 68690 68869 36.36
ARS606 VI 167606 168041 18.65
30
Table 3. Numbers of origins in different quartiles.
Table 4. Sequencing read counts of rDNA region.
WT rpd3Δ sir2Δ sir2Δrpd3Δ
Averaged
rDNA Counts 231455 544027 186044 385527
Averaged
Total Counts 4215311 10184800 3943919 6835320
rDNA/Total
Ratio 0.0550 0.0534 0.0476 0.0564
Standard
Deviation 0.0006 0.0005 0.0018 0.0007
1st-
quartile
2nd-
quartile
3rd-
quartile
4th-
quartile
total
(number/percentage)
WT 123 58 26 28 235
52.34% 24.68% 11.06% 11.91% 100.00%
rpd3∆ 130 90 43 38 301
43.19% 29.90% 14.29% 12.62% 100.00%
sir2∆ 108 38 21 25 192
56.25% 19.79% 10.94% 13.02% 100.00%
sir2∆rpd3∆ 123 54 21 28 226
54.42% 23.89% 9.29% 12.39% 100.00%
31
Table 5. Total sequencing read counts.
WT rpd3∆ sir2∆ sir2∆rpd3∆
Single-Copy DNA
Counts 150500 166265 71738 117840
rDNA Counts 32015 5247 67208 70231
Total Counts 182515 171512 138946 188071
Absolute %rDNA 17.54% 3.06% 48.37% 37.34%
Absolute %single-copy 82.46% 96.94% 51.63% 62.66%
%rDNA to WT 82.46% 91.10% 39.31% 64.56%
%single-copy to WT 17.54% 2.87% 36.82% 38.48%
Total 100.00% 93.97% 76.13% 103.04%
32
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Abstract (if available)
Abstract
In budding yeast Saccharomyces cerevisiae, the exact mechanism that regulates DNA replication timing remains unclear. Previous studies have reported that two histone deacetylases Rpd3 and Sir2 control replication initiation. In this study, we investigated Rpd3 and Sir2 in regulation of replication timing genome-wide using qBrdU-IP-seq. Our results revealed that Rpd3 delays initiation at many of the single-copy late-firing origins by direct acting on them while Sir2 significantly represses initiation at rDNA origins and controls single-copy origins timing in an indirect manner.
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University of Southern California Dissertations and Theses
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Asset Metadata
Creator
He, Yiwei
(author)
Core Title
The role of Rpd3 and Sir2 in regulation of replication initiation in budding yeast: Rpd3 acts directly on single-copy origins while Sir2 works through ribosomal DNA origins
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Publication Date
09/23/2016
Defense Date
08/15/2016
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
Fkh1,OAI-PMH Harvest,qBrdU-IP-Seq,rDNA,replication timing,Rpd3,Sir2
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Frenkel, Baruch (
committee chair
), Aparicio, Oscar (
committee member
), Rice, Judd (
committee member
)
Creator Email
yiweihe@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c40-304598
Unique identifier
UC11279627
Identifier
etd-HeYiwei-4800.pdf (filename),usctheses-c40-304598 (legacy record id)
Legacy Identifier
etd-HeYiwei-4800.pdf
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304598
Document Type
Thesis
Format
application/pdf (imt)
Rights
He, Yiwei
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
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The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
Fkh1
qBrdU-IP-Seq
rDNA
replication timing
Rpd3
Sir2